1. Field of the Invention
In triple-negative breast cancer (TNBC) there is recurrent genetic alteration of pathway components. Using shRNA methods, we observed that the zinc finger transcription factor Kruppel-like factor 4 (KLF4) can promote RAS-ERK signaling in TNBC cells. Endogenous KLF4 bound to the promoter regions and promoted the expression of two microRNAs (miRs), miR-206 and miR-21 (miR-206/21). Antisense-mediated knockdown (anti-miR) revealed that miR-206/21 coordinately promote RAS-ERK signaling and the corresponding cell phenotypes by inhibiting translation of the pathway suppressors RASA1 and SPRED1. The present invention identifies RASA1 and SPRED1 mRNAs as latent RAS-ERK pathway suppressors that can be upregulated in tumor cells by anti-miR treatment. Consequently, KLF4-regulated miRs are important for the maintenance of RAS-ERK pathway activity in TNBC cells. The present invention provides a method of treating a patient having cancer comprising providing an upregulation of RASA1 and SPRED1 by anti-microRNA (anti-miR) treatment. The pronounced inhibitory effect of anti-miR-206/21 on the level of activated ERK 1/2 identifies the enhanced translation of RASA1 and SPRED1 as an attractive therapeutic modality. Further, this invention provides suppression of KLF4 or the anti-sense mediated silencing of miR-206 and/or miR-21 to be used in combination with MEK inhibitors or other pathway antagonists to attenuate drug resistance in cancer patients.
2. Background of the Invention
In comparison to simpler organisms, the evolution of metazoans required adaptations for the proper regulation of cell fate (1). One such adaptation is the mitogen-activated protein kinase (MAPK) pathway composed of RAS, RAF, MEK, and ERK, which regulates a variety of cell physiologic processes (2-9). Diverse stimuli including growth factors, interaction with extracellular components, and cell stress can signal through receptor tyrosine kinases (RTKs), integrins, or ion channels to regulate signaling through the RAS GTPases. GTP-bound RAS (RAS-GTP) can activate MAP3Ks (i.e., the RAF family of protein kinases), leading to sequential phosphorylation and activation of MAP2Ks (i.e., MEK 1/2) and the extracellular signal-regulated kinases (ERK 1/2).
Inhibitory proteins play important roles in RAS-ERK pathway regulation. These include the RAS p21 protein activator (GTPase activating protein [GAP]) 1 (RASA1), the GAP neurofibromin 1 (NF1), the sprouty homologs SPRY1 and SPRY2, and the sprouty-related, EVH1 domain containing (SPRED) proteins, SPRED1 and SPRED2 (10-12). SPRED1 associates with NF1 to mediate its membrane localization, implicating GAP activity as a shared molecular mechanism among pathway inhibitory proteins (13). Congenital disorders that deregulate this kinase cascade include Neurofibromatosis type I, Legius syndrome, Noonan syndrome, Costello syndrome, and cardiofaciocutaneous syndrome (8,9,14-16).
In addition, somatic alteration of this pathway is critical for the initiation and progression of a variety of cancers. Activating point mutation of RAS genes or BRAF occur in approximately 15-30% and 7% of all human cancers, respectively (3,17-20). In human breast cancer, point mutation of these genes is rare, but activated ERK 1/2 levels are frequently elevated and contribute to the aggressive behavior of cancer cells (21,22).
RAS-ERK pathway activity appears particularly critical in triple-negative breast cancers (TNBCs), tumors that are deficient in estrogen receptor (ER) a, HER2, and progesterone receptor (23,24). This group of clinically aggressive tumors overlaps extensively with the basal-like and claudin-low molecular subtypes (25). Genomic analysis of human basal-like breast tumors indicates frequent copy number gain of KRAS (32%) and BRAF (30%), and reduced gene copy number for pathway inhibitors such as RASA1 and DUSP4 (26-31). For RASA1, the correlation of mRNA levels, genomic copy number and clinical outcome supports a functional role in TNBC (29). Consistent with these results, basal-like breast tumors have a high RAS-ERK pathway activity signature (24,30). Despite this insight, therapeutic targeting of the pathway is hindered by cellular mechanisms of escape, including dynamic reprogramming of the kinome and PI 3-kinase activation, and improved strategies for inhibiting the pathway are needed (32,33).
The zinc-finger transcription factor Kruppel-like factor 4 (KLF4) is a pluripotency factor that functions in tumors in a context-dependent fashion, capable of exerting both pro-tumorigenic and anti-tumorigenic effects (34-36). Supporting a tumor suppressor role, its expression is reduced during development of tumors such as colorectal cancer, and endogenous Klf4 suppresses tumorigenesis in the ApcMin mouse model (37). In normal cells, KLF4 is often induced in response to cell stress or wounding, and protumorigenic influences may reflect its role in the stress response (38-46). Loss- or gain-of-function studies show that KLF4 can promote malignant properties, including epithelial transformation in vitro, escape from RAS-induced senescence, enhanced cell survival following γ-radiation-induced DNA damage, increased tumorigenicity of colorectal cancer stem cell-enriched spheroid cells, and skin tumor initiation in transgenic mice (43,47-50).
In human breast cancers, there is typically higher expression of KLF4 in tumor cells compared with the adjacent, uninvolved epithelium. This elevated protein level, or else demethylation of the KLF4 promoter, portends a poor prognosis (51-54). We previously identified microRNA (miR)-206 as a potential downstream effector of KLF4 that, in turn, directly regulates KLF4 translation, constituting a feedback loop (55). miRs associate with the RNA-induced silencing complex (RISC) to regulate the translation of cognate mRNAs. miR deregulation occurs in multiple cancer types and can promote or inhibit tumorigenesis (56-58).